Over the past twenty years, the method of choice for the chemical synthesis of oligonucleotides (ONs) has been the phosphoramidite four-step process which utilizes the reaction of deoxynucleoside phosphoramidites with a solid phase tethered nucleoside or oligonucleotide (Letsinger, R. L.; Lunsford, W. B. J. Am. Chem. Soc. 1976, 98, 3655-3661; Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-1862; Matteucci, M. D.; Caruthers, M. H. J. Am. Chem. Soc. 1981, 103, 3186-3191).
Initially the 5′-O-dimethoxytrityl (DMT) group is removed from a deoxynucleoside linked to the polymer support. Step 2, elongation of a growing oligodeoxynucleotide, occurs via the initial formation of a phosphite triester intemucleotide bond. This reaction product is first treated with a capping agent designed to esterify failure sequences and cleave phosphite reaction products on the heterocyclic bases. The nascent phosphite intemucleotide linkage is then oxidized to the corresponding phosphotriester. In the final step of each cycle, the DMT group is removed from the growing oligonucleotide using a large excess of a weak acid, trichloroacetic acid (TCA), in an organic solvent. Further repetitions of this four-step process generate the ON of desired length and sequence. The final product is cleaved from the solid phase and obtained free of base and the β-cyanoethylphosphate (Sinha, N. D.; Biernat, J.; Koster, H., Tetrahedron Lett. 1983, 24, 5843-5846) protecting groups by treatment of the support with concentrated ammonium hydroxide, methyl amine or other nucleophillic strong bases (Ogilvie, K. K.; Theriault, N. Y.; Seifert, J-M.; Pon, R. T.; Nemer, J. J. Can. J Chem. 1980, 58, 2686-26930).
We have recently developed a new method of deprotection of oligonucleotides that does not require strong bases like ammonia or methyl amine. This method utilizes the strong nucleophilicity of peroxyanions at mildly basic pH. This is especially applicable to the chemical synthesis of oligoribonucleotides (RNA). Since this method has many significant advantages for the deprotection of oligonucleotides, it is especially appropriate to develop protecting groups that are specifically designed to utilize these novel deprotection conditions. Although many of the standard protecting groups in the prior art can be removed using these novel conditions, those standard protecting groups were optimized for removal using strong bases. In addition, several of the standard protecting groups require strong bases. A clear example of this is the β-cyanoethylphosphate protecting group. This group is typically removed by a β-elimination reaction (see U.S. Pat. Re34,069 to Koster et al.). The typical β-elimination reaction occurs by having an electron withdrawing group in the α-position to a methylene carbon. This makes the protons of the α-methylene group acidic and they can thereby be removed using a strong base. The compound then eliminates the phosphate in the β-position and forms an alkene such as acryonitrile.

Although this works well for the use of a strong base like ammonia, we typically use peroxyanion solutions at pH conditions below 11. At this pH the proton cannot easily be abstracted, and these β-elminination reactions are not well suited for use with peroxyanions.
The use of strongly electron withdrawing groups such as the cyanoethyl groups has the additional disadvantage of deactivating the phosphoramidite reagent toward coupling of the intemucleotide bond. This is especially important in the chemical synthesis of RNA. In typical RNA synthesis the 2′-hydroxyl is protected creating additional inhibition of coupling by crowding around the reactive phosphorus center.
As shown above, protonation by azole acid catalysts followed by nitrogen exchange converts the phosphite species to several highly active electrophiles. An electron withdrawing protecting group can significantly decrease the reactivity of the active intermediates. This inhibition is made worse by the crowding around the active phosphorus reagent that occurs in the chemical synthesis of RNA as a result of the protected 2′-hydroxyl (—OR in the following scheme).

While there are examples of phosphorus protecting groups in the literature, there remains a need for novel phosphorus protecting groups for polynucleotides, e.g. for use during polynucleotide synthesis.